T‐cell responses in COVID‐19 survivors 6−8 months after infection: A longitudinal cohort study in Pune

Abstract Background The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) immune response is crucial for disease management, although diminishing immunity raises the possibility of reinfection. Methods We examined the immunological response to SARS‐CoV‐2 in a cohort of convalescent COVID‐19 patients in matched samples collected at 1 and 6−8 months after infection. The peripheral blood mononuclear cells were isolated from enrolled study participants and flow cytometry analysis was done to assess the lymphocyte subsets of naive, effector, central memory, and effector memory CD4+ or CD8+ T cells in COVID‐19 patients at 1 and 6−8 months after infection. Immunophenotypic characterization of immune cell subsets was performed on individuals who were followed longitudinally for 1 month (n = 44) and 6−8 months (n = 25) after recovery from COVID infection. Results We observed that CD4 +T cells in hospitalized SARS‐CoV‐2 patients tended to decrease, whereas CD8+ T cells steadily recovered after 1 month, while there was a sustained increase in the population of effector T cells and effector memory T cells. Furthermore, COVID‐19 patients showed persistently low B cells and a small increase in the NK cell population. Conclusion Our findings show that T cell responses were maintained at 6−8 months after infection. This opens new pathways for further research into the long‐term effects in COVID‐19 immunopathogenesis.


| INTRODUCTION
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that led to the coronavirus disease in 2019 (COVID- 19) is still a hazard to the general public's health.Since the beginning of the pandemic, there have been more than 5.3 million recorded deaths and over 275 million confirmed cases (WHO weekly epidemiological update on COVID-19, December 21, 2021).The predominant symptom of COVID-19 is a respiratory illness with symptoms ranging from asymptomatic or moderate infection to severe symptoms necessitating intensive care unit (ICU) hospitalization. 1 To control and eradicate viral infections, a unique adaptive immune response must be developed.More specifically, virus-specific T and B cells are stimulated, grow, and eventually develop into effector cells in response to infection.Neutralizing antibodies and memory B and T cells, which are specific to the viral antigen survive long after the infection has been eradicated.This memory immune response, which is activated during vaccination, is crucial in the prevention of reinfection.To comprehend the emergence and persistence of such protective immunity, it is crucial to characterize in detail the extent of specific adaptive immune responses in convalescent COVID-19 patients with varying degrees of severity.For foreseeing and controlling potential future waves of infections in the general population, a deeper understanding of the mechanisms driving the development of protective immunological memory in recovered individuals is of paramount importance for public health.
The early response to SARS-CoV-2 infection in severely ill COVID-19 patients is marked by significant immunological dysfunctions linked to a systemic inflammatory response and the emergence of altered innate and adaptive immune responses. 1,2More particular, T cell response is significantly altered in critically ill COVID-19 patients, and the most severe COVID-19 patients have been reported as having severe lymphopenia, phenotypic, and functional T cell alterations. 3Therefore, it is still uncertain whether these critically ill patients can develop a strong and long-lasting SARS-CoV-2 specific T cell response despite the presence of significant immunological changes during the stay in the hospital.
Considering this, the objective of the current investigation was to monitor the immunological response, including memory T cells specific to SARS-CoV-2, in samples obtained 1 and 6-8 months after infection from a cohort of convalescent critically ill COVID-19 patients.COVID-19 patients were identified through a combination of epidemiological history, clinical symptoms, and whose nasal/throat swab positive test result by SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) as per the WHO Interim Guidance. 4Patients with COVID-19 were included in the study upon hospitalization.The initial blood samples for immunological investigations were collected around the 5th day of COVID-19 infection, with an average deviation of ±2 days, and two subsequent samples were collected at 1 and 6−8 months, respectively, measured from the time of enrollment in the study.Twenty-one healthy individuals were recruited as uninfected controls for immunophenotype comparison.All uninfected controls had no history of SARS-CoV-2 infection during the previous 6 months and were confirmed negative by COVID-19 RT PCR test.The study was carried out in compliance with good clinical practices, including the International Conference on Harmonization Guidelines and the Declaration of Helsinki.

| Patient characteristics
The COVID-19 patients were administered an approved questionnaire upon enrollment to collect information regarding COVID-19-related symptoms before hospitalization and smoking behaviors.The date of COVID-19 diagnosis, highest level of care received, the maximum amount of oxygen supplementation required (unit, oxygen need 5 L/min supplemented by High Flow Nasal Oxygen or Continuous Positive Airway Pressure, and the critical care provided (ICU, with or without mechanical ventilation) for COVID-19 illness were all gathered from digital medical records.The patients with severe/moderate illness were those who required clinical care and required oxygen support.In brief, the moderate patients had oxygen saturation (SpO 2 ) of 90%−94%, mostly required supplement oxygen of 2−6 L and did not require noninvasive ventilation.The severe patients had oxygen saturation (SpO 2 ) of ≤90%, mostly required supplement oxygen of 4−20 L and noninvasive ventilation or highflow oxygen.

| Peripheral blood mononuclear cells (PBMC) isolation
EDTA anticoagulated peripheral blood (5 mL) was collected from each individual.All samples were tested within 6 h of blood collection.The PBMCs and plasma were separated by density gradient centrifugation according to standard protocols and the plasma was stored at −80°C till further use.

| Detection of anti-SARS-CoV-2 antibodies
Plasma samples were assessed for the presence of anti-SARS-CoV-2-specific binding antibodies using the commercially available Abbott SARS-CoV-2 IgG assay (M/s Abbott Laboratories' Diagnostics Division) and conducted on the ARCHITECT machine as per the manufacturer's instructions.Test results were quantified as anti-SARS-CoV-2 index values, where an index >1.4 was interpreted as a positive result.

| Statistical analysis
SPSS (Statistical Package for Social Sciences) version 26.0;IBM was used to analyze the data.The Shapiro-Wilk test was used to assess the distribution of the data set.Total counts (frequency), percentages, means, and standard deviations were generated as part of descriptive statistics for patient demographics.For continuous variables, an independent sample t-test/Mann-Whitney U test was employed, and for categorical connections, a χ 2 or Fisher exact test was utilized.Spearman's rank correlation was used for the correlation analysis, and a p value of ≤.05 was considered statistically significant.

| Clinical characteristics of COVID-19 patients
During an 18-month period (March 2021 to September 2022), blood samples were collected from hospitalized COVID-19 patients.A subset of donors was followed longitudinally at 1 month (n = 44) and up to 6-8 months (n = 25).Our analysis focused on the effect of COVID-19 on various T cell subsets and B cell immune responses.The clinical and demographic characteristics of the hospitalized COVID-19 cohort are summarized in Table 1.Our hospitalized COVID-19 cohort represents the infection in wider society in terms of biological sex distribution, encompassing 25 males and 19 females, and a mean age of 51.4 years (Table 1).Out of the total participants, only 12 patients took their COVID-19 vaccination during the study period, while the remaining individuals did not receive the vaccine.
Table 2 provides the biochemical laboratory parameters for hospitalized COVID-19 patients at enrollment in the study.The disease categorization for the patients were moderate or severe as per the guidelines given by the National Institutes of Health. 5

| Hospitalized SARS-CoV-2 patients had a persistent decline in CD4+ T cells, whereas CD8+ T cells gradually recover after 1 month
To assess the longitudinal effect of COVID-19 on T cell populations, we sampled PBMCs from patients with COVID-19 over a period of 6−8 months.PBMCs were analyzed by flow cytometry to investigate immunophenotype across the various T cell compartments.
At baseline, lymphocyte counts were notably lower (p = .03)compared to those of healthy controls.However, the lymphocyte populations recovered, with a substantial increase in cell counts at 1 month (p = .0003)that remained consistent until 6−8 month (p = .0003)reaching levels comparable to those of healthy controls (Figure 1A).Similarly, total T cells (CD3+ T cells) were significantly diminished at baseline (p = .0001)compared to healthy controls.Total T cells (CD3+ T cells) increased significantly after 1 month (p < .0001)and then dropped slightly at 6−8 months (p = .022).Nonetheless, T cell counts remained significantly higher at 1 and at 6−8 months than baseline (p = .021)and were comparable to healthy controls (Figure 1B) (Table 3).
When comparing CD4+ T cells, the percentages at baseline were comparable to healthy controls, however, a constant and significant reduction was observed at 1 month (p = .022)which is continued until 6−8 months (p = .0016)as compared to the healthy controls and baseline (Figure 1C).In relation to CD8+ T cells, the increase was significantly higher at 6−8 months compared to healthy controls (p < .0001),at baseline (p = .017),and at 1 month (p = .012)(Figure 1D) (Table 3). 8][9][10]

| COVID-19 patients have a sustained, elevated effectors T cells and effector memory T cells population
The environment established by an infection, both locally and systemically, can cause changes in general memory T cell populations. 11We looked at how COVID-19 affected naive, effector, and memory CD4+ and CD8+ T cells.Over time, distinct variations in the CD4+ and CD8+ T populations of COVID-19 patients were detected (Table 3).The fraction of CD4+ naive T cells and CD4+ central memory cells decreased significantly with time when compared to healthy controls and at baseline (Figure 1E,G).At 6−8 months after discharge from hospital, there was a considerable increase in the CD4+ effectors and CD4+ effector memory T cell fractions compared to results obtained at the time of hospitalization and healthy controls (Figure 1F,H).We observed that CD8+ T cells showed more sustained decrease in their naive and central memory as compared to healthy controls and from baseline to 6−8 months after enrollment (Figure 2A,C).The frequencies of CD8+ effectors and CD8+ T effector memory, on the other hand, increased from baseline to 6−8 months (Figure 2B,D) and also in comparison to healthy controls.Overall, we found alterations in the memory T cell pool, most notably in the CD4+ and CD8+ effector memory subsets, where the changes persisted until the end of the study.

| Patients with COVID-19 had a persistently low B cells and minimal increase in NK cell population
We further assessed B cells, NK cells, and NKT cells in peripheral blood to better understand lymphocyte attrition.The cytotoxic and regulatory fractions of NK cells increased slightly but not significantly with time compared to baseline and healthy controls (Figure 2E,F).There was a substantial decline in B cells from 1 month (p = .043)to 6−8 months (p = .0034)when compared to hospitalization and healthy controls (Figure 2G).Despite this, no significant changes in NKT cells were observed over time (Figure 2H).

| Comparison of immune cell subsets between patients with moderate and severe conditions
We further analyzed the differences in immune cell subsets in moderately and severely ill patients.We observed that lymphocytes and CD3+ T cells were increase over a period in both moderate and severely ill patients (Figure 3A-D).In case of CD4+ T cells, the   percentages decreased significantly over a time while remained comparable in severe cases (Figure 3E,F).
When comparing CD8+ T cells, the percentages were declined at 1 month and then recovered over a period of 6−8 months in both the cases (Figure 3G,H).The CD4+ T cell memory populations, CD4+ naïve T cells and CD4+ central memory cells decreased over a period of time while CD4+ effectors and CD4+ effector memory T cells increased over a span of 6−8 months from baseline in both moderate and severe cases (Figure 3I,J).In CD8+ T cell memory populations, CD8+ naïve T cells and CD8+ central memory cells decreased over a period of time while CD8+ effectors and CD8+ effector memory T cells increased over a span of 6−8 months from baseline in both moderate and severe cases (Figure 4A,B).
We further assessed B cells, NK cells, and NKT cells in moderated and severe cases.The cytotoxic (Figure 4C,D) and regulatory fractions (Figure 4E,F) of NK cells increased slightly but not significantly with time compared to baseline in both moderate and severe cases.However, no significant changes in NKT cells were observed over time in both cases (Figure 4G,H).When compared to baseline, there was a decrease in B cells from 1 to 6−8 months in both cases (Figure 4I,J) (Table 4).

| Paired analysis of immune subsets in COVID-19 patients at baseline and 6 months
Further we have analyzed 25 COVID-19 patients paired data to address any bias due to follow-up.We have found that percentages of lymphocytes, CD3 + T cells, CD4+ effectors, CD4+ effector memory T cells, CD8+ effectors, CD8+ effector memory T cells, and NK regulatory cells were significantly increased while CD4+ naïve, CD4+ central memory, CD8+ naïve, CD8+ central memory T cells, and B cells were significantly decreased at 6−8 months as compared to the baseline.However the percentages of CD4+ T cells, CD8+ T cells, NKT cells, and NK cytotoxic cells remain comparable at baseline and 6−8 months (Figure 5).

| Analysis of T cell subsets based on age and gender in COVID-19 patients
To further investigate the impact of demographic differences on T cell proportions as the age and gender can influence the percentages of T cells in the study.In  5).

| Anti-SARS-CoV-2 antibodies shows a decline over a period of 6−8 months in COVID-19 patients
We investigated the antibody index for the SARS-CoV-2 in COVID-19 patients over a span of 6−8 months.We found that the anti-SARS-CoV-2 antibody index is significantly increased after 1 month (median: 5.26; range: 0.3−10) as compared to the baseline (median: 1.12; range: 0.01−10.22)which is further significantly decreased over a period of 6−8 months (median: 1.26; range: 0.19−10.06)and remain comparable to baseline (Figure 6G).

| DISCUSSION
In our longitudinal study cohort, we utilized spectral flow cytometry to track T cell populations throughout the course of COVID-19, from initial inclusion to 6−8 months postrecovery.Our findings revealed extensive modifications within the T cell compartment, notably an increase in effector and effector memory T cells persisting for 6−8 months posthospital discharge.Lymphopenia, a common observation in COVID-19 patients upon hospitalization, [12][13][14][15][16] may result from T cells relocating or undergoing cell death during this stage of the disease.8][19][20] Our research, along with others,' indicates that the T cell compartment is disproportionately affected compared to B or NK cells, [21][22][23][24] underscoring the pivotal role of T cell subsets in COVID-19 pathogenesis.Additionally, reductions in circulating NKT cells, as demonstrated by our study and others, 25 have been associated with severe COVID-19 disease and unfavorable outcomes. 26,279][30] Long-term studies, spanning 8−9 months, are relatively limited. 31vidence from the 2003 SARS-CoV outbreak indicates that anti-SARS-CoV antibodies declined to undetectable levels within 2 years, 32 while SARS-CoV-specific memory T cells remained detectable even 11 years after the outbreak. 33Memory T cells represent a crucial and varied subset of T cells that retain antigen experience over the long term. 34They can be mobilized into effector cells upon reinfection or exposure. 35Depending on their cellular programming and phenotype, they are categorized into various central and effector memory subtypes.Among these, the effector subsets include CD45RA + CCR7− TEMRA cells, which essentially consist of effector memory T cells (TEM) that reexpress CD45RA following antigen stimulation.While the functionality of this population remains somewhat elusive, CD4 + TEM-RA cells have been implicated in contributing to protective immunity. 36oreover, heightened levels of virus-specific CD8+ effector cells persist following dengue vaccination. 37In our study, we observed a sustained elevation of both CD4+ and CD8+ effector T cells, as well as effector memory T cells, throughout the 6−8 month duration, which contrasts with previous findings where CD4+ effector levels remained unaffected by COVID-19. 38revious research has demonstrated an increase in the CD8 + TEMRA population upon hospitalization, 39,40 maintained for up to 6 weeks. 39The precise role of CD8+ effectors in COVID-19 remains largely uncertain, although Cohen et al. 31 noted an increase in SARS-CoV-2-specific CD8+ effector cells over time.In our investigation, we examined the entire expanded CD8+ effector and CD8+ effector memory T cell population, without confirming if there was a larger fraction of antigenexposed, that is, SARS-CoV-2-specific, T cells within the cohort.
Our research revealed that all COVID-19 patients exhibited an increase in effector T cells and effector memory T cells, which continued to rise over time.These findings align with other studies demonstrating that SARS-CoV-2 infection leads to heightened expansion of antigen-specific CD4+ and CD8 + T cell subsets. 41,42owever, it remains uncertain whether the lower antigen-specific responses observed at 1 month compared to 6−8 months are attributable to overall immunosuppression 43 or a natural progression of the immune response over time. 44urrently, all immunocompetent individuals develop SARS-CoV-2-specific antibodies, a trend evident in our study as well.While some research indicates that these antibodies are detectable for only a few months postinfection, 45,46 others suggest their persistence for at least 6 months. 47,48In our cohort, we observed that the increase in B cells endured for 6−8 months postinfection, despite experiencing a significant decline at some point after 6 weeks.Our findings align with those of Björkander et al., 42 who reported that antibody responses lasted up to 8 months in young adults.Even as antibody levels may decline, as observed in our study, affinity maturation continues, potentially enhancing the potency of SARS-CoV-2-specific humoral immunity to provide protection against severe disease and increase the ability to neutralize the virus. 491][52] Persistent symptoms such as fatigue, cognitive impairment, and respiratory issues may reflect ongoing immune dysregulation or inflammation.Chronic activation of the immune system could lead to exhaustion of immune cells, impaired functionality, or autoimmunity.Additionally, the presence of viral reservoirs or lingering viral particles may sustain immune activation, contributing to prolonged inflammation and tissue damage.Understanding these interactions between long-term symptoms and immune responses is crucial for developing effective therapies and interventions for individuals with persistent COVID-19 symptoms.
Our study focused on moderate and severe COVDI-19 patients, the observed immune responses likely have relevance to milder or asymptomatic cases as well.The findings suggest that even in milder or asymptomatic cases of COVID-19, T cell responses may play a crucial role in providing long-term immunity.This indicates that the immune system mounts a robust response to the virus, potentially conferring lasting protection against reinfection.Further research into the immune dynamics of milder cases is warranted to fully understand the implications for long-term immunity and disease management.
Our study examined various immune cells and their subsets in COVID-19 patients, yet we did not conduct an in-depth analysis of regulatory T cells.This represents one of the limitation of our research.
Despite the formidable challenges posed by the pandemic, we believe that the cohort examined in this investigation is well characterized and offers significant value.This study sheds light on the shifts in immune response seen during hospital-treated SARS-CoV-2 infection and recovery.Our longitudinal data reveal profound alterations in the T cell landscape persisting for over 6−8 months.These findings, alongside others, contribute crucial insights into SARS-CoV-2 cellular and humoral immunity, paving the way for further exploration and enhanced comprehension of the long-term immunopathogenic changes observed in COVID-19.
Study designThe present study was approved by the Institutional Ethics Committee of Dr D. Y. Patil Vidyapeeth (Ref No-DYPV/EC/634/2021 dated February 25, 2021) and carried out between March 2021 to February 2022 at Dr. D. Y. Patil Medical College, Hospital and Research Centre, Pune.The participants were ≥18-years-old male and female subjects who provided written informed consent before enrollment.

F
I G U R E 1 Flow cytometry analysis of T cells subsets.PBMCs were stained and acquired on flow cytometer.The bar graphs represent the comparison of percentages of immune cells and their subpopulation in healthy controls and COVID-19 patients at different time point (A) lymphocytes, (B) CD3+ T cell, (C) CD4+ T cells, (D) CD8+ T cells, (E) CD4+ naïve T cells, (F) CD4+ effectors T cells, (G) CD4+ central memory T cells, and (H) CD4+ effector memory T cells profile.Data are represented as median with range.*p < .05,**p < .001,and ***p < .0001significant.HC, healthy controls; PBMC, peripheral blood mononuclear cells.

T A B L E 3
Percentages of different immune cell subsets in COVID-19 patients at different time points.

F I G U R E 2
Flow cytometry analysis of CD8+ T cells, NK, and B cell subsets.The bar graphs represent the comparison of percentages of immune cells and their subpopulation in healthy controls and COVID-19 patients at different time points (A) CD8+ naïve T cells, (B) CD8+ effectors T cells, (C) CD8+ central memory T cells, (D) CD8+ effector memory T cells, (E) NK cytotoxic cells, (F) NK regulatory cells, (G) B cells, and (H) NKT cells profile.Data are represented as median with range.*p < .05,**p < .001,and ***p < .0001significant.F I G U R E 3 Flow cytometry analysis oimmune cell subsets in moderate and severe COVID-19 cases.The bar graphs represent the comparison of percentages of immune cells and their subpopulation at different time point in moderate and severe COVID-19 cases, respectively (A, B) lymphocytes, (C, D) CD3+ T cells, (E, F) CD4+ T cells, (G, H) CD8+ T cells, and (I, J) CD4+ memory T cells profile.Data are represented as median with range.*p < .05,**p < .001,and ***p < .0001significant.

F
I G U R E 4 Flow cytometry analysis of CD8 T cells, B cells, and NK cells subsets in moderate and severe COVID-19 cases.The bar graphs represent the comparison of percentages of immune cells and their subpopulation at different time point in moderate and severe COVID-19 cases, respectively (A, B) CD8 + T cell memory subsets, (C, D) NK cytotoxic cells, (E, F) NK regulatory cells, (G, H) NKT cells, and (I, J) B cells.Data are represented as median with range.*p < .05,**p < .001,and *** p < .0001significant.T A B L E 4 Comparison of percentages of different immune cell subsets in moderate and severe cases.Moderate Severe Baseline 1 Month follow-up 6 Month follow-up Baseline 1 Month follow-up 6 Month follow-up Lymphocytes 33.6 (7.06−63.2memoryT cells (CD8 + CD45RA-CCR7-CD62L−) case of age we divided the patients into three groups 18−40 years, 41−60 years, and above 60 years.We found that as the age increases the percentages of lymphocytes, CD4 + T cells and CD8 + T cells decreases over a span of 6−8 months from baseline (Figure 6A−C).However in case of gender, the percentages of lymphocytes, CD4 + T cells, and CD8 + T cells remains comparable in male and females over a span of baseline to 6−8 months (Figure 6D−F) (Table

F I G U R E 6
Flow cytometry analysis of T cell proportions on basis of age and gender and SARS-CoV-2 IgG status for COVID-19 patients.(A−C) The bar diagram shows comparison of percentages of lymphocytes, CD4 + T cells, CD8 + T cells, respectively in COVID-19 patients with age groups 18−40 years, 41−60 years, and above 60 years.(D−F) The bar diagram shows comparison of percentages of lymphocytes, CD4 + T cells, CD8 + T cells, respectively in COVID-19 infected males and females.(G) The whiskers box plot shows the anti-SARS-CoV-2 antibody index in COVID-19 patients at baseline, 1 month, and after 6−8 months.The red line indicates cut off value.Data are represented as median with range.*p < .05,**p < .001,and ***p < .0001significant.
Demographic and clinical characteristics of the enrolled study participants.
T A B L E 1Abbreviation: NA, not applicable.